Non-Homologous End-Joining (NHEJ) is a DNA double strand break repair pathway essential in maintaining the integrity of the genome. NHEJ requires the DNA Ligase IV/XRCC4 complex and the DNA-dependent Protein Kinase (DNA-PK), which is formed by DNA-PKcs, and the heterodimer Ku70/80. Inositol hexakisphosphate was shown to stimulate NHEJ in vitro, however, in vitro studies also suggest that NHEJ can be further stimulated by one or more additional unidentified factors. This research project will investigate the participation of an additional stimulatory factor in mammalian NHEJ. First, the unidentified stimulatory activity will be isolated by fractionation of human whole cell Extracts. Second, candidate stimulatory factors will be identified using protein sequencing and database searches, and participation of each candidate stimulatory factor will be confirmed utilizing the in vitro end-joining assay. Finally, the mechanism of NHEJ stimulation by the discovered factor will be explored. Results from this research will help understand the mechanism of NHEJ in vivo. This will facilitate the development of therapies for people with mutated NHEJ genes, and also anticancer treatments that can target NHEJ factors to sensitize cells before radiotherapy.